LOCUS       sequence-256391-        4729 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Piggybac vector for SRIRACCHA-mediated mutation enrichment (donor 
            repair template for HDR).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4729)
  AUTHORS   Wen Y, Liao G, Pritchard T, Zhao TT, Connelly JP, Pruett-Miller SM, 
            Blanc V, Davidson NO, Madison BB
  TITLE     A stable but reversible integrated surrogate reporter for assaying 
            CRISPR/Cas9-stimulated homology-directed repair.
  JOURNAL   J Biol Chem. 2017 Apr 14;292(15):6148-6162. doi: 
            10.1074/jbc.M117.777722. Epub 2017 Feb 22.
  PUBMED    28228480
REFERENCE   2  (bases 1 to 4729)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1074/jbc.M117.777722"; journalName: "J Biol Chem"; date: 
            "2017-04-14- 14"; volume: "292"; issue: "15"; pages: "6148-6162"
COMMENT     Sequence Label: pBS-PtH
FEATURES             Location/Qualifiers
     source          1..4729
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    13..34
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(49..54,55..72,73..79)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    87..103
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     92..114
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     111..127
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     111..127
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     146..166
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        148..166
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     195..211
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     196..212
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             870..923
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             930..1952
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             930..1952
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     primer_bind     complement(2062..2081)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2063..2081)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2091..2108)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2091..2107)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2100..2122)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2248..2703)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2385..2406)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2597..2616
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2730..2834
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2835..2903,2904..3695)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3053..3072)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3866..4454
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4355..4374
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4608..4625
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
       61 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
      121 ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctggg
      181 taccgggccc cccctcgagg tcgacggtat cgataagctt gcatgcctgc aggtcggccg
      241 ccacgaccgg tgccaccatg actgaataca aaccaactgt tcgcctggca actcgtgatg
      301 atgttccacg tgcagttcgc accctggctg ctgcatttgc tgactaccct gcaacccgtc
      361 acactgtgga cccagaccgc cacattgaac gtgtgactga actgcaggag ctgttcctga
      421 cccgtgtggg cctggacatt ggcaaagtgt gggtggcaga tgatggtgct gctgtggcag
      481 tgtggaccac ccctgaatct gttgaagctg gtgcagtgtt tgctgagatt ggcccacgca
      541 tggcagaact gtctggcagc cgcctggcag cacaacagca gatggaaggt ctgctggcac
      601 cacaccgccc aaaagaacct gcttggttcc tggcaactgt gggtgtgagc cctgaccacc
      661 agggtaaggg cctgggctct gcagtggtgc tgcctggtgt ggaagcagct gaacgtgcag
      721 gtgtgcctgc tttcctggag acctcagctc cacgcaacct gcctttctat gaacgcctgg
      781 gcttcactgt gactgctgat gtggaagtgc cagaaggccc acgcacttgg tgcatgactc
      841 gcaaaccagg tgctagcctt ggctccggcg agggcagggg aagtcttcta acatgcgggg
      901 acgtggagga aaatcccggc ccactcggca aaaagcctga actcaccgcg acgtctgtcg
      961 agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg
     1021 aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata
     1081 gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc
     1141 tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct
     1201 cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc
     1261 tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg
     1321 ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat
     1381 gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg
     1441 cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc
     1501 ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa
     1561 cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca
     1621 tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga
     1681 ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg
     1741 accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc
     1801 gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca
     1861 gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac
     1921 gccccagcac tcgtccgagg gcaaaggaat agagcggccg cgactccagt tcgtggccgt
     1981 cgatcgttta aagggaggta gtgagtcgag atatcgatac tagttctaga gcggccgcca
     2041 ccgcggtgga gctccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc
     2101 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     2161 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     2221 cagttgcgca gcctgaatgg cgaatggaaa ttgtaagcgt taatattttg ttaaaattcg
     2281 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     2341 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     2401 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     2461 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     2521 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     2581 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     2641 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     2701 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2761 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     2821 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     2881 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     2941 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     3001 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     3061 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     3121 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     3181 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     3241 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     3301 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     3361 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     3421 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     3481 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     3541 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     3601 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     3661 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     3721 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     3781 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     3841 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     3901 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     3961 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     4021 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     4081 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     4141 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     4201 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     4261 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     4321 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     4381 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     4441 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     4501 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     4561 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     4621 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     4681 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtga
//
